CYP2S1 is a recently identified human cytochrome P450, expressed extensively in epithelial tissues. We propose that CYP2S1 plays a significant role in the metabolic activation of environmental procarcinogens, and the metabolism of pharmaceuticals and endogenous compounds. The proposal will address this hypothesis and characterize regulation of the enzyme. There are three specific aims: (i) We have expressed human CYP2S1 in bacteria, and demonstrated that it metabolizes several compounds that are toxic and/or carcinogenic to epithelial tissues. We will also over-express the enzyme in mammalian cells. Using these expression systems, we will screen for additional substrates, and also for procarcinogens that are activated to mutagenic (and therefore probably carcinogenic) derivatives by CYP2S1. The Km and Vmax values will be determined for representative compounds, and the metabolites that are formed will be identified. The degree to which CYP2S1 contributes towards the total metabolism of particular substrates in human epithelial tissues will be determined using an inhibitory antibody to the enzyme, (ii) We have shown that CYP2S1 is inducible by dioxin, carcinogenic polycyclic aromatic hydrocarbons (PAHs), and hypoxia. We will investigate whether the potential Xenobiotic Responsive Elements (XREs), or the potential Antioxidant Response Element (ARE) in the 5'flanking region of the human CYP2S1 gene mediate induction by dioxin and/or PAHs, and address the hypothesis that due to the particular nucleotide sequences of the above XREs, the gene responds better to PAHs than to dioxin in certain cells. We will also analyze the mechanism of hypoxic induction of the gene, (iii) We will generate a knockout mouse for Cyp2s1, and then generate a derivative of this mouse containing the human CYP2S1 gene, including its flanking regulatory regions. This """"""""CYP2S1-humanized"""""""" mouse will be used to study the metabolism of substrates of human CYP2S1, the biological consequences of this metabolism, and the regulation of the human CYP2S1 gene by xenobiotics and hypoxia, thus complementing and extending specific aims 1 and 2. Our studies may demonstrate important roles for CYP2S1 in the metabolism of carcinogens, Pharmaceuticals and endogenous compounds, and may ultimately provide opportunities for reducing the deleterious effects of environmental carcinogens and the adverse effects of certain Pharmaceuticals in the human population.
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